The processes involved in the assembly of complex, multimolecular biological structures are poorly understood. This project seeks to use the striated muscle myofibril as a model system in which to explore these mechanisms. Extremely large myofibrillar proteins are hypothesized to play a key role in myofibril assembly. Therefore, as a first step to studying the function of extremely large myofibrillar proteins using molecular genetic approaches, we are cloning, sequencing, and expressing portions of mouse nebulin, as well as N-RAP, a nebulin-related muscle specific protein. Southern blot data along with previously determined sequence data suggest that nebulin and N-RAP sequences are highly conserved among vertebrate species, underlining their functional importance to muscle function. In the past year, we developed a protocol for purifying expressed nebulin fragments and incorporating them into actin filaments in vitro. Using this novel assay, we found that a 28 module nebulin fragment binds actin filaments with a stoichiometry of one nebulin module per actin monomer, while a 19 module fragment binds with a stoichiometry of one nebulin module per two actin monomers. Our data suggests a ratio equivalent to one nebulin module per actin monomer in vivo. The results show that we can incorporate the longer nebulin fragment into actin filaments to the same extent found in vivo, and suggest that shorter nebulin fragments may not be correctly incorporated into thin filements in vitro. We completed the cDNA sequencing of N-RAP, a novel nebulin-related protein. The full length N-RAP cDNA contains an open reading frame of 3525 base pairs which is predicted to encode a protein of 133kDa. A 587 amino acid region near the C-terminus is 45% identical to the actin binding region of human nebulin, containing more than two complete 245 residue nebulin super repeats. The N-terminus contains the consensus sequence of a cysteine rich LIM domain, which may function in mediating protein-protein interactions. These data suggest that the encoded protein may link actin filaments to some other protein or structure. A gel overlay assay verifies that N-RAP can bind actin filaments in vitro. We have also obtained an antibody that binds cloned N-RAP and specifically labels about 180 kDa band on immunoblots of skeletal and cardiac muscle. Using this specific antibody, we found that N-RAP is located at the myotendon junction in skeletal muscle and at the intercalated disc in cardiac muscle. We are testing the hypothesis that N-RAP serves as a vital mechanical link between the terminal myofibrils and the extracellular matrix. Future studies will explore the physiological functions of nebulin and the nebulin-related family of actin binding proteins.